PROJECTSUMMARY/ABSTRACT Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States. Strong geneticdriversoftumorigenesis,suchaslossoffunctionoftheAPCtumor-suppressorgene,havebeenwell characterized,buttheroleofepigeneticeventsremainspoorlyunderstood.Mousemodelsofintestinalcancer have shown that DNA methylation is critical for tumor formation, but it is not known which DNA methylation events are required for tumorigenesis. Both human and mouse intestinal tumors display extensive promoter hypermethylation, and a striking number of these hypermethylated genes are normally occupied by the Polycomb Repressive Complex in stem cells. Polycomb Target Genes (PTGs) are classically known for their important regulatory role in stem cell differentiation. Therefore, based on the established importance of DNA methylationinintestinaltumorigenesis,thehighfrequencyofaberrantstem-cellPTGmethylationinCRC,and theknownbiologyofPTGs,wehypothesizethataccumulationofPTGDNAhypermethylationinintestinalstem cells (ISCs) potentiates tumor formation through interference with normal stem cell differentiation. Stem cells withadifferentiationimpedimentcouldgraduallyexpandintoapoolofself-renewing,proliferatingcellsthatare unable to terminally differentiate and shed like normal, short-lived differentiating intestinal cells. An abnormal expansionofthestemcellcompartmentwouldproportionatelyincreasetheincidenceofdrivergeneticevents, andthestem-likepropertiesconferredbyimpededdifferentiationmayenhancethemalignanttransformationof such cells. To test our hypothesis, we will use intestinal organoid cultures to pursue the following specific aims:(1)DeterminetheconsequenceofPTGDNAhypermethylationonISCdifferentiation,and(2)Determine theroleofPTGinactivationinintestinaltumorigenesis.InAim1,wewillestablishanovelmodelofPTGDNA hypermethylation by knocking out Kdm2b, which protects PTGs from de novo methylation, in intestinal organoids. This system will allow us to visualize and measure the differentiation capacity of ISCs containing widespreadPTGDNAhypermethylation.InAim2,wewillbuildupontheKdm2bmodeltodeterminetheeffect of such hypermethylation on the proliferation and spheroid-forming capacity of Apc-deficient organoids. Further, we have prioritized candidate PTG drivers of intestinal tumorigenesis using bioinformatic analysis of PTG DNA hypermethylation in intestinal tumors. The top candidates will be functionally validated using a CRISPR/Cas9knockoutapproachinorganoidculturefollowedbytheaforementionedtumorigenicityassays. The long-term objectives of this project are to understand the role of PTG DNA hypermethylation in intestinaltumorigenesisandtoidentifyPTGsthatcouldserveastherapeutictargetsforCRCbytranscriptional reactivation using focused epigenetic therapy. Given the extensive PTG DNA methylation events found in human CRC and the demonstrated role of DNA methylation in tumorigenesis, our investigation of PTG hypermethylationmayyieldnewinsightsintointestinaltumorigenesisandintopotentialepigenetictherapies.